Animal model

The study was performed on sixty-four 5–6 months old C57BL/6 J mice were divided into four groups i.e. Control group (21 % O2, 24 h per day, 8 weeks, n = 16); Hypoxia group (Intermittent hypoxia: 7 % O2 60 s and alternating 20 % O2 60 s, 8 h per day, 8 weeks, n = 16); Hypoxia + 90RC and Hypoxia + 270RC group (Intermittent hypoxia for 1st 4 weeks; Intermittent hypoxia pretreated 90 mg/Kg and 270 mg/Kg Rhodiola Crenulata by oral gavage per day for 2nd 4 weeks, each n = 16). After exposure with normoxia or hypoxia and saline or RC, the hearts were excised and analyzed by heart weight index, H&E staining, TUNEL-positive assays and Western Blotting. Ambient temperature was kept at 25 °C and the animals were exposed to an artificial 12-h light–dark cycle, the light period which began at 7:00 am. Mice were provided with standard laboratory meals (Lab Diet 5001; PMI Nutrition International Inc., Brentwood, MO, USA) and water ad libitum. Protocols were proved by the Institutional Animal Care and Use Committee of China Medical University, Taichung, Taiwan.

Rhodiola crenulata

Rhodiola Crenulata was provided by TCM Biotech International Corp (Taiwan). The extraction process was as follows: The raw material was analyzed and verified to be free of organic toxins and heavy metals (Pb, Hg, Cd, As, Cu) or below acceptable levels. The raw material was ground and sifted with a 20 mesh screen. The grainy powder was extracted and compressed then freeze dried to reduce water content to less than 8 %. The material was then ground to return to a powder form (water less 6 %). The powder was sifted then packaged in a sterile aluminum bag.

Echocardiography

The trans-thoracic echocardiographic images of all mice were obtained using Philips M2424A ultrasound systems (Andover, MA) in anesthesia with 1 % isoflurane through a nose cone. The M-mode echocardiographic inspection was executed applying a 6–15 MHz linear transducer (15–6 L) through a parasternal long axis approach. The left ventricular M-mode dimensions on the stage of the papillary muscles contained inter-ventricular septum (IVS), left ventricular internal end-diastolic dimensions (LVIDd), left ventricular internal end-systolic dimensions (LVIDs), posterior wall thicknesses (LVPW), and fractional shortening (FS). FS% was calculated as a result of the equation FS% = [(LVIDd-LVIDs)/LVIDd]/100.

Cardiac characteristics

The hearts of mice in the four groups were extracted and cleaned with PBS. The weights of the left ventricles and the whole heart were measured. Once weighting, the 6 hearts of mice in each group were soaked in formalin and added analyzed by Hematoxylin-eosin, Masson trichrome staining, DAPI staining and TUNEL assay. The other 6 hearts of mice in each group were cleaned, frozen, and added analyzed by Western Blotting. Besides, right tibia lengths were measured using a digital caliper and the left heart weight were weighed. The whole heart weight (WHW) to body weight (BW) ratio, the left ventricle weight (LVW) to body weight (BW) ratio, the left ventricle weight (LVW) to the to the whole heart weight (WHW) ratio, and the whole heart weight to tibia length and the left ventricle weight to tibia length ratio were determined for analysis.

Tissue extraction

The left ventricle samples of cardiac tissue extracts were homogenized in a lysis buffer at a ratio of 100 mg tissue/1 ml buffer for 1 min to obtain cardiac tissue extracts. The homogenates were put on ice for 10 min then centrifuged at 12,000 g for 40 min twice. The supernatant was collected and stored at −70 °C.

H&E staining, Masson trichrome staining, and TUNEL

The heart was extracted then soaked in formalin, then dehydrated via graded alcohols, after that embedded in paraffin wax. In heart samples, the 0.2-μm thick paraffin sections were cut from paraffin-embedded tissue blocks. The tissue samples were dewaxed using in xylene immersion, and then rehydrated. For Hematoxylin-eosin staining (H&E staining), the slices were dyed with hematoxylin and eosin. For Masson Trichrome Staining, the slices were dyed with Masson Trichrome. After lightly rinsing with water, the slides were dehydrated through graded alcohols then immersed in Xylene twice. Photomicrographs were obtained by Zeiss Axiophot microscopes. The Terminal Deoxynucleotide Transferase-mediated dUTP Nick End Labeling (TUNEL) assay were studied with an apoptosis detection kit (Roche Applied Science, Indianapolis, IN, USA) and observed that TUNEL-positive nuclei (fragmented DNA) fluoresced bright green at 450–500 nm. The mean number of TUNEL-positive cells were calculated for 5–6 individual fields x 2 slices x 3 regions of the left ventricle (the upper, the middle, the lower) removed from 6 mice hearts within all groups. All data counts were executed in two independent individuals within a blind study.

Statistical analysis

The weight index, protein levels and percentage of TUNEL positive cells were compared with the Control, the Hypoxia, the Hypoxia + RC90 and the Hypoxia + RC270 groups by analysis of variance with pre-planned contrast comparison. In every case P < 0.05 was considered significant.

Body weight and cardiac characteristics

Body weight, whole heart weight, left ventricular weight in the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 groups were similar to the Control group (Table 1). Whole heart weight (WHW), left ventricular weight (LVW), whole heart weight normalized by tibia length (WHW/Tibia), left ventricular weight normalized by tibia length (LVW/Tibia) in the Hypoxia + RC90, and the Hypoxia + RC270 groups were similar to the Control group. Inter-ventricular septum at diastole (IVSd), left ventricle posterior wall thicknesses (LVPW), left ventricle internal dimension at diastole (LVIDd), left ventricular internal end-systolic dimensions (LVIDs) in the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 groups were similar to the Control group (Table 1). Compared to the Control Group, fractional shortening (FS %) was decreased by chronic intermittent hypoxia about 6.2 %. Compared to the Hypoxia Group, the Fractional Shortening (FS %) was increased in the Hypoxia + RC90 group (4.8 %) and Hypoxia + RC270 group (8.0 %), respectively.

Cardiac histopathological changes of left ventricle

To determine if there were changes in cardiac architecture, a histopathological analysis of ventricular tissue stained with hematoxylin and eosin was performed. We viewed 400X magnified images and found that the ventricular myocardium in the Control group showed normal architecture and interstitial space. We found abnormal myocardial architecture and the increased interstitial space in the Hypoxia group. These myocardial architecture abnormalities in the Hypoxia + RC90, Hypoxia + RC270 groups were less than those in the Hypoxia group (Fig. 1a).

Fig. 1

a Representative histopathological analysis of cardiac sections from left ventricles in the Control, the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 RC groups was performed with Hematoxylin and eosin (H&E) staining and Masson trichrome staining method. The images were magnified by 400 times. b The Representative stained apoptotic cells of cardiac sections from left ventricles in the Control, the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 RC groups were measured by TUNEL assay with dark background (upper panels, green spots) and staining with DAPI (lower panels, blue spots). The images were magnified by 400 times. c Bars presents the percentage of TUNEL positive cells relative to the total DAPI cells (n = 6 in each group). **P < 0.01, denotes significant differences from the Control group. ##P < 0.01 denotes significant differences from the Hypoxia group

TUNEL-positive apoptotic cells of left ventricle

To analyze the apoptotic activity in cardiac tissues, the apoptotic cells and total cells were measured by TUNEL assay and DAPI staining respectively. Hypoxia groups stained with TUNEL assay had the most TUNEL-positive cardiac cells when compared to the Control group, Hypoxia + RC90 or the Hypoxia + RC270 groups. Decreases in the number of TUNEL-positive cardiac cells were detected in the Hypoxia + RC90 and the Hypoxia + RC270 groups compared with the Hypoxia group. TUNEL-positive cells relative to DAPI-stained nuclei cells in the hypoxia group about 4.1 % is higher than those in the Hypoxia + RC90 and the Hypoxia + RC270 group both about 0.9 % (Fig. 1).

To determine the upstream components of cardiac Fas receptor dependent apoptotic signaling pathways in mice with treatment of RC under chronic intermittent hypoxia, protein levels of Fas receptor and FADD in the excised hearts of the Control, the Hypoxia, the Hypoxia + RC90 and the Hypoxia + RC270 groups were examined by Western blotting. Compared with the Control group, Fas receptor and FADD were significantly increased in the Hypoxia group (Fig. 2). Fas receptor and FADD in the Hypoxia + RC90 and Hypoxia + RC270 groups were significantly lower than those in the Hypoxia group (Fig. 2).

Fig. 2

a The representative protein products of Fas receptor and Fas-associated death domain (FADD) extracted from the left ventricles of excised hearts in the Control, the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 RC groups were measured by Western Blot analysis. b The bars represent the relative protein quantification of Fas receptor and Fas-associated death domain (FADD) on the basis of α-tubulin, respectively, and indicates Mean values ± SD (n = 6 in each group). **P < 0.01, ***P < 0.001, are the significant differences from the Control. ###P < 0.001, are the significant differences from the Hypoxia group

To evaluate the cardiac Bcl-2 family in mitochondria-dependent apoptotic pathways in mice with chronic intermittent hypoxia and treatment with RC, we analyzed the protein levels of the Bcl-2 family (Bcl-xL, Bcl-2, Bax, Bad, p-Bad) in the excised hearts of the Control group, the Hypoxia, the Hypoxia + RC90 and Hypoxia + RC270 groups by Western Blotting. Mitochondrial related anti-apoptotic protein of Bcl-xL, Bcl-2, p-Bad in the Hypoxia + RC90 and Hypoxia + RC270 groups were higher than those in the Hypoxia group. Mitochondrial related pro-apoptotic proteins of Bax and Bad were much higher in the Hypoxia group than the Control group as well as the Hypoxia + RC90 and Hypoxia + RC270 groups (Fig. 3). The protein level of pro-apoptotic t-Bid, a main intracellular molecule signaling mediator from Fas to mitochondrial pathway, in the Hypoxia + RC90 and Hypoxia + RC270 groups were significantly lower than the protein in the Hypoxia group (Fig. 3).

Fig 3

a The representative protein products of Bcl-xL, Bcl2, Bax, p-Bad, Bad and t-Bid extracted from the left ventricles of excised hearts in the Control, the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 RC groups were measured by Western Blotting analysis. b The bars represent the relative protein quantification of Bcl-xL, Bcl2, Bax, p-Bad, Bad and t-Bid on the basis of α-tubulin and indicates Mean values ± SD (n = 6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, are the, significant differences from the Control. #P < 0.05, ##P < 0.01, ###P < 0.001are the significant differences from the Hypoxia group

To realize the downstream components of cardiac Fas receptor (caspase 8 and 3) and mitochondrial (caspase 9 and 3) dependent apoptotic pathways, the caspase 8, 9 and 3 were measured by Western blotting in hearts excised from the four experimental groups. Western blot analysis showed that the protein products of activated caspase 8, 9, and 3 were higher in the Hypoxia groups than the Control group. The protein level of activated caspase 8, 9, and 3 in the Hypoxia + RC90 and Hypoxia + RC270 groups were much lower than in the Hypoxia group (Fig. 4).

Fig. 4

a The representative protein products of activated caspase 8, activated caspase 9, and activated caspase 3 extracted from the left ventricles of excised hearts in the Control, the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 RC groups were measured by Western Blotting analysis. b The bars represent the relative protein quantification of activated caspase 8, activated caspase 9, and activated caspase 3 on the basis of α-tubulin, respectively, and indicates Mean values ± SD (n = 6 in each group). **P < 0.01, ***P < 0.001 are the significant differences from the Control. ##P < 0.01, ###P < 0.001, are the significant differences from the Hypoxia group

Cardiac VEGF-related pro-survival pathway

To understand the VEGF-related pro-survival pathway in mice with chronic intermittent hypoxia and treatment with RC, the VEGF, p-PI3k, p-Akt were measured by Western blotting in hearts excised from the four experimental groups. Western blot analysis showed that the protein products of VEGF, p-PI3k and p-Akt were higher in the RC groups than the Control group (Fig. 5). The protein levels of VEGF, p-PI3k and p-Akt in the Hypoxia + RC90 and Hypoxia + RC270 groups were much higher than those in the Hypoxia group (Fig. 5).

Fig. 5

a The representative protein products of activated enhance VEGF-related pro-survival pathway (VEGF, p-PI3k, p-AKT) cardiac pro-survival pathway extracted from the left ventricles of excised hearts in the Control, the Hypoxia, the Hypoxia + RC90, and the Hypoxia + RC270 RC groups were measured by Western Blotting analysis. b The bars represent the relative protein quantification of VEGF, p-PI3k, and p-AKT on the basis of α-tubulin, respectively, and indicates Mean values ± SD (n = 6 in each group). **P < 0.01, ***P < 0.001 are the significant differences from the Control. ##P < 0.01, ###P < 0.001, are the significant differences from the Hypoxia group

Experimental design

In humans, Rhodiola Crenulata extract treatment of 800 mg daily for 7 days before ascent and 2 days during mountaineering of Hehuan Mountain in Taiwan prevented the headaches of Acute Mountain Sickness [31]. The dosage of Rhodiola Crenulata powder 100 mg/Kg and 500 mg/Kg, once daily for 4 weeks, ameliorate sucrose-induced acute hyperglycemia by gavage in mice [32]. Our current study of treatment with Rhodiola Crenulata in mice was designed as 90 mg/Kg and 270 mg/Kg by oral gavage. Our study suggests that the current dosage did protect chronic intermittent hypoxia-induced cardiac widely dispersed apoptosis. The reasons why long-term intermittent hypoxia was designed as 8 weeks were based on our previous studies. Long-term intermittent hypoxia planned for 8 weeks could cause abnormal myocardial architecture and increased pro-apoptotic BNIP3 and Bad proteins on rat’s hearts [21] as well as could induce cardiac widely dispersed apoptosis [5, 22]. Therefore, our study design of 8 weeks chronic intermittent hypoxia as negative control group and Rhodiola Crenulata treated for 4 weeks was validated based on the current findings. Besides, we should caution that any Rhodiola Crenulata-induced anti-apoptotic effect in the present investigation might be caused by various factors and cannot be isolated to any specific mechanism, such as effects of salidroside, effects of tyrosol, anti-oxidation, inhibiting free radicals, enhancing stem cells, inducing erythropientin or other unclear factors.

Our study suggests that the protein levels of VEGF-related pro-survival pathway (VEGF, p-PI3k, p-AKT) were decreased after chronic intermittent hypoxia on mice hearts as well as Rhodiola Crenulata prevented chronic intermittent hypoxia-induced cardiac widely dispersed apoptosis. Salidroside was found to drive effective antioxidant properties and protect cells from apoptosis by the PI3K/Akt pathway [35]. In our previous study, salidroside had cardiac protection through chronic intermittent hypoxia-induced apoptosis [5]. In two studies, salidroside protected against hypoxia-induced cardiomyocytes necrosis and apoptosis by increasing VEGF levels [36], as well as increased Akt phosphorylation, cardiomyocytes against injury by PI3K/Akt pathway and increased PI3K antioxidant enzyme [37]. Salidroside and Tyrosol, two isolated compounds of Rhodiola, prevented apoptosis in H9c2 cells, reduced caspase-3 activity, reduced cytochrome c release and showed the anti-apoptotic effect of the combination of the two components was more beneficial than that of salidroside and tyrosol independently [38].

Acknowledgement

The study is supported by the National Science Council (NSC 98-2622-B-039-004-CC3), Asia University (CMU98-asia-08) and China Medical University (CMU99-COL-21), Taiwan. This study supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002). We’d like to thank TCM Biotech International Corp., Taiwan for providing Rhodiola Crenulata in this study.

Competing interests

All authors declare that they have no competing interests.

Authors’ contributions

M-C L raises the idea of the current studies, participated in the coordination of studies and drafted the manuscript. P-Y P, Y-M L, Y-L Y, S-M C and Y-F L help in animal models, experimental processes and echocardiographic images. M-H L performed the statistical analysis. S-D L, C-Y H and J-G L participated in the study design, funding and coordination. All authors read and approved the final manuscript.

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